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Background: Iron induced heart failure is the commonest cause of death in thalssaemia major. The cardiomyopathy is reversible when treated early, but once symptomatic heart failure occurs, the outlook is poor. A recent technique using magnetic resonance myocardial T2* (T2 star) has been validated for the assessment of early myocardial iron deposition. Worldwide however, transthoracic echocardiography is the mainstay of assessment for these patients. Conventional echo parameters are poor at identifying those with cardiac iron loading who are at risk of cardiac failure, only becoming abnormal once significant iron loading has occurred. Newer techniques involving tissue Doppler imaging (TDI) have been promising. Our aim was to explore the role of these techniques and that of brain natriuretic peptide (BNP), a biomarker released in heart failure, in the management of Thalassaemia major patients. Methods/Results: 167 patients with thalassaemia major were screened with cardiac T2* MRI to quantify iron loading. Those with severe cardiac iron, T2* <8ms, (n=15) were treated with both desferrioxamine and deferiprone chelation. Those with mild to moderate cardiac iron, T2* 8- 20ms, (n=65) were randomised to desferrioxamine plus either deferiprone or placebo. Both groups were treated for 12 months. At baseline, 6 and 12 months all those in the combination therapy trial and in the severe group were assessed with T2*, full transthoracic echocardiography including TDI and BNP levels. BNP only became abnormal in those with obvious clinical heart failure. TDI systolic velocities in the septum and RV were significantly lower in those with severe cardiac iron and improved over 12 months, correlating with an improvement in T2*. Conclusion: In thalassaemia patients, BNP is unhelpful in assessing cardiac iron status. Systolic tissue velocities can give an indication as to an individuals cardiac iron status and could help to monitor their progress during chelation therapy.

Enterococcus faecium is an opportunistic human nosocomial pathogen that has developed resistance to many existing antimicrobial therapies. Treatment of enterococcal infections is becoming increasingly challenging and there are limited therapeutic options against multidrug-resistant enterococci. The work described in this thesis aimed to identify genes involved in resistance to host stresses and virulence as these could potentially be future therapeutic targets. An E. faecium transposon mutant library was screened for altered resistance to lysozyme and to nisin as a model antimicrobial peptide. This approach led to the identification of several genes that contribute to lysozyme and nisin resistance. A number of mutants that were sensitive to nisin had a transposon insertion in a gene predicted to encode a tyrosine decarboxylase. It was shown that the tyrosine decarboxylase plays a role in acid tolerance and mediates virulence of E. faecium in a Galleria mellonella larvae infection model. A targeted genetic approach was used to examine the role of an E. faecium serine threonine protein kinase (Stk1) in antimicrobials resistance, host cell stresses and virulence. Disruption of the stk1 gene led to higher sensitivity to antibiotics that target the penicillinbinding proteins and bile salts compared to the wild type. Providing the stk1 gene from either E. faecium or ireK of E. faecalis in trans restored the resistance of the E. faecium stk1 mutant to ceftriaxone. The stk1 gene also contributed to E. faecium virulence in the G. mellonella infection model. Through bioinformatic analysis a putative ATP-binding-cassette (ABC) transporter system with the potential to be involved in antimicrobial peptide resistance was identified. Markerless deletion of the gene encoding the permease component of the ABC transporter reduced E. faecium resistance to the antimicrobial peptide nisin and altered the net charge of the bacterial cell surface. Genes involved in acid tolerance, antimicrobial resistance and virulence of E. faecium were identified in this study.

Staphylococcus aureus is well-known for its ability to acquire resistance to a broad range of antimicrobial agents and a limited number of commercially available antibiotics exist that are active against multidrug resistant strains. Antimicrobial peptides have been suggested as promising alternatives to current antimicrobials due to their potent antimicrobial activity against a broad range of microorganisms including multidrug resistant bacteria, and a membrane-lytic mode of action that is thought to have low possibility of inducing bacterial resistance. This study describes the identification of S. aureus genes involved in resistance to the human cationic antimicrobial peptide LL-37, with a particular interest in the effects of a physiological concentration of bicarbonate on the resistance mechanism. Transposon mutagenesis and recombinase-based in vivo expression technology systems were designed to enable genome-wide screening. A S. aureus transposon mutant library was screened for increased resistance to LL-37 in the presence of bicarbonate. Mutants with insertions in yycH and yycI, demonstrated bicarbonate-dependent resistance to LL-37. Both yycH and yycI form part of a predicted operon yycFGHI in S. aureus, and have been shown to be suppressors of an essential two component system YycFG in B. subtilis that regulates cell wall metabolism. The resistance of S. aureus small colony variants (SCVs) to LL-37 was also investigated. SCVs defective in hemB, menD or aroD, demonstrated bicarbonate-dependent resistance to LL-37. Furthermore, SigB (a global regulator) and TcaR (an activator of protein A) were found to exert opposite effects on resistance to LL-37 in the presence of bicarbonate. Strains defective in TcaR showed bicarbonate-dependent resistance to LL-37, interestingly, this resistance was abolished by either deleting sigB or repairing tcaR in these strains. These data suggest that YycFG, SigB, TcaR and the SCV phenotype may play important roles in resistance to LL-37 under in vivo conditions where bicarbonate is present.